β-Amyloid accumulation in aged canine brain: A model of early plaque formation in Alzheimer's disease

0197-4580/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.

Neurobiology of Aging, Vol. 14, pp. 547-560, 1993 Printed in the U.S.A. All fights reserved.

13-Amyloid Accumulation in Aged Canine Brain: A Model of Early Plaque Formation in Alzheimer's Disease B R I A N J. C U M M I N G S , .1 J O S E P H H. S U , * C A R L W. C O T M A N , * R U S S E L L W H I T E ~ A N D M I C H A E L J. R U S S E L L $

*Department of Psychobiology, University of California, Irvine, CA 92717-4550 7~Departments of LEHR and ~:Anesthesiology, University of California, Davis, CA 95817 Received 23 October 1992; Revised 12 M a y 1993; Accepted 16 June 1993 CUMMINGS, B. J., J. H. SU, C. W. COTMAN, R. WHITE AND M. J. RUSSELL. f3-Amyloidaccumulationin agedcaninebrain: A modelof earlyplaqueformation in Alzheimer'sdisease. NEUROBIOL AGING 14(6) 547-560, 1993.--We characterized eight aged beagles (maintained from birth in a laboratory colony) and one black Labrador using Bielschowsky's, thioflavine S, and Congo red staining, and antibodies to the 13-amyloidpeptide, dystrophic neurites, and other plaque components. All plaques within these canine brains were of the diffuse subtype and were neither thioflavine S- nor Congo red-positive. The majority of plaques in the entorhinal cortex contained numerous neurons within them while plaques in the dentate gyms did not. 13-Amyloid immunoreactivity was also present within select neurons and neuronal processes and was detected as a diffuse linear zone corresponding to the terminal fields of the perforant path. There was no significant correlation between extent of [3-amyloid accumulation and neuron number in entorhinal cortex. Neither tau-1, PHF-I, nor SMI-31-immunostaining revealed dystrophic fibers, confirming the classification of these plaques as diffuse. Canine plaques did not appear to contain bFGF- or HS-positive immunostaining. This may explain why neuritic involvement was not detected within these canine plaques. It is possible that the 13-amyloid within the canine brain has a unique primary structure or may not be in an assembly state that adversely affects neurons. Alzheimer's disease Plaque formation

Animal model 13-Amyloid Trophicfactor Cerebellum Canine Neuropathology

ANIMAL models of Alzheimer's disease (AD) pathology are needed to elucidate the mechanisms of amyloid accumulation and plaque formation in AD. Primate models of AD have been well described, at both the light and electron microscopic level. Plaques have been found in the aged rhesus monkey, squirrel monkey, and orangutan (42). Primates exhibit all plaque subtypes found in humarts but do not have neurofibrillary tangles (NFTs) (47,55). It appears that the density and plaque subtypes in primates reflect those in nondemented older humans as compared to AD (47). However, primate tissue can be as difficult to obtain as human tissue, and needs to be 20 or 30 years of age to show these changes. Most recently, neuritic plaques, dystrophic neurites, and alterations in pyramidal neurons have been seen in the aged lemur, a primate with a shorter lifespan (8-12 years) than the rhesus monkey (2). Another possible alternative to model AD may be through the use of a canine model, which has long been recognized as exhibiting some characteristics of AD neuropathology (4,14, 30,54). Regardless of the species, plaques result from the deposition of 13-amyloid, which is derived from the 13-amyloid precursor protein (13-APP). 13-APP mRNA has been detected in canine brain (1), and

Neuritic dystrophy

Neuronalcounts

the conservation of the 13-APP sequence in canine was recently confirmed by Johnstone et al., in 1991 (22). Thus, it is likely that, as in the human brain, 13-APP in the canine brain is also processed to form 13-amyloid. In light of recent cell culture work, however, it is unclear whether processing of 13-APP into 13-amyloid is abnormal or more common than previously believed (6,19). A variety of reports on the neuropathology present within the aged canine have been presented. However, discrepancies exist concerning the type and extent of AD-like pathology in canines. For example, Uchida et al. reported both primitive and neuritic plaques in three of nine aged dogs of mixed breed upon staining with both Bielschowsky and Congo red. These dogs also had amyloid angiopathy with cerebral hemorrhage (50). Conversely, Giaccone et al. report plaques in five of seven aged dogs of mixed breeds with immunostaining with 13-amyloid antibodies; they did not detect plaques with either thioflavine S or Congo red staining (16). Thus, while AD-like pathology has been found in canine material, the extent and type of pathology needs clarification. The appearance of discrepancies in the literature may result from the breed of dog examined, the environment in which the dogs were raised, or the age of the dog. These potentially confounding fac-

To whom requests for reprints should be addressed. 547

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CUMMINGS ET AL.

tors could be overcome by studying a well-defined population of dogs. In addition to using a defined subject group, the study of canine pathology should more completely examine potential similarities and differences between pathology observed in aged canine tissue and AD tissue. For example, it would be desirable to verify if dystrophic neurites exist within canine plaques. Also, recent findings show that trophic factors such as basic fibroblast growth factor (bFGF) (17,46) and neurite substrate factors such as heparan sulfate glycosaminoglycans (HSGAGs) (44,48) colocalize with some AD plaques. We have suggested that these molecules may serve to misdirect neuronal processes into plaques where they become dystrophic (8,10). The presence or absence of these molecules in the plaques of the canine brain relative to neuritic involvement may provide a suitable model for the study of these plasticity mechanisms. In order to address these issues, we examined a group of kennel-mate beagles. These animals were raised in a controlled setting, maintained in good health, and have available medical records (39). We examined the brains of these beagles with Bielschowsky, thioflavine S, and Congo red staining, and with immunocytochemistry using antibodies to 13-amyloid (1328 and [342) and neuritic markers (phosphorylated neurofilament, SMI-31; tau- 1; and paired helical filament, PHF-1). The presence of astrocytic involvement in pathology was also assessed by immunostaining with antibodies against glial fibrillary acidic protein (GFAP). In addition, because a subset of AD plaques contain bFGF or HSGAG's, we investigated the locus of these two antigens in canine tissue. Lastly, if the plaques found in the canine brain are, indeed, at an early stage of development, they should be similar to plaques found in the human cerebellum, a region where few neuritic plaques are found (26). Thus, we compared canine plaques to human cerebellar plaques. METHOD Canine Tissue

The brains from eight well-characterized aged beagles and one 17-year-old black Labrador retriever were examined (see Table 1). The black Labrador was a community raised animal requiring euthanasia due to senescence. For this study, primarily the hippocampus and entorhinal cortex were used. The beagles were control animals from a United States Department of Energy (DOE) lowlevel radiation/longevity study (39). The group of beagles examined here received no radiation exposure. All necessary procedures were taken to promote their long-term survival and monitor their health, including quarterly examinations of major organ systems and annual physical exams by a veterinarian (including EKG, fecal analysis for parasites, and a whole-body radiographic scan). Animals were housed in an outdoor kennel, fed once daily, and given water ad lib. The mean age of these control beagles was 16.0 years, significantly greater than the 6-year average lifespan of excess litter mates who were donated to be raised in the community. Dogs died spontaneously or were euthanized when terminally ill. Their brains were kept in 10% neutral-buffered formalin and archived for future DOE use. Upon our receipt, canine brains were transferred to phosphatebuffered saline, pH 7.4 (PBS). Serial sections (50 In,m) were cut on a vibratome, collected free floating in PBS, and processed for immunohistochemistry, modified Bielschowsky's silver stain (57), thioflavine S, and Congo red staining (38). Neighboring sections were also stained for cresyl violet to visualize the general cytoarchitecture of the areas under study and for cell counting.

TABLE 1 AGE, SEX, AND PRIMARYCAUSEOF DEATHOR DIAGNOSISFOR CANINES AND HUMANSUSEDIN THIS STUDY ID #

Sex

Age

Primary Cause of Death--Diagnosis

ROOM54 ROOM55 ROOX42

male male female

14.9 16.3 17.3

ROOF48

female

17.1

DOOF24

female

15.0

ROOY41

male

i 6.5

ROOY39

male

13.3

ROOM50 Black Lab

male male

17.3 17"

Lymphocarcinoma Perforatingesophageal ulcer Heart disease, myelopathy, intervertebrial disk disease Myelopathy, intervertebral disk disease, some evidence of heart disease Heart disease, metastatic tubular adenocarcinoma-mammary gland Congestive heart failure, evidence of heart disease Heart disease, metastatic, malignant oral melanoma Renal carcinoma Senescence,elective euthanasia

UCI 40-91 UCI 62-91 UCI 69-91 UCI 76-91 UCI 47-90 UCI 46-9t UCI 57-91

female male female male male female male

72 79 86 79 91 71 83

"Definite" AD (CERAD) "Definite" AD (CERAD) "Definite" AD (CERAD) "Definite" AD (CERAD) "Definite" AD (CERAD) Normal with respect to dementia Normal with respect to dementia

All canines were DOE beagles, except the black Labrador* which was raised in the community and estimated to be 17 years old. Human cases, coded UCt were from the IRU in brain aging at University of California, Irvine. Human Tissue

All AD tissue (n -- 5) was from neuropathologically defined cases, and met the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) criteria for the pathological diagnosis of AD (29). Control cases (n --- 2) were matched for age as closely as possible (see Table 1). Human tissue was prepared as previously described (12). Immunohistochernistry

All antibodies used in this study and their dilutions are listed in Table 2. For immunohistochemistry, tissue sections were first treated for 20 min with 3.0% H2Oz, 10% methanol in PBS to inactivate endogenous peroxidase activity. Sections were incubated overnight at room temperature (23°C) in anti-j328 (1:4000) or anti-1342 (1:1000). These 13-amyloid markers are affinity purified rabbit polyclonal antibodies directed against amino acids 1-28 or 1--42 of the 13-amyloid peptide, respectively (12,48). Sections were then incubated in biotinylated goat antirabbit IgG (Vector, CA) and avidin-biotin complex for 1 h each. The final color product for single labeling was visualized by a diaminobenzidine (DAB) kit (Vector, CA) for a brown reaction product. In the case of double-labeling experiments, benzidine dihydrochloride (BDHC) was used (24). For doubIe labeling, sections were rinsed after the DAB reaction and then incubated overnight in a pan-neuronal marker, SMI-311 (Sternberger Monoclonals, MD). Sections were rinsed, then incubated in biotinylated horse antimouse IgG and avidin-biotin complex for 1 h each. Sections were then rinsed (5 x) in 0.01 M sodium phosphate buffer (PB, pH 6.8), and preincubated in 0.01% BDHC, 0.025% sodium nitroferricyanide in PB and reacted by the addition of 0.005% H20 z to fresh

AMYLOID ACCUMULATION IN AGED CANINE BRAIN

549

TABLE 2 ANTIBODIESUSEDIN THIS STUDY, INCLUDINGTHE ABBREVIATIONUSEDIN THE TEXT, WHATIS DETECTEDBY EACHANTIBODY, THE DILUTIONUSED, AND THE SOURCE Antibody

Dilution

Source

GFAP HSGAG bFGFtyr~ i Tau-I PHF- 1

13-amyloidpeptide(1-28) ~-amyloid peptide(l-42) Astrocytes Heparan sulfate glycosaminoglycans Basic fibroblast growth factor Abnormally phosphorylated tau protein Paired helical filaments-tau

1:4000 1:1000 1:5000 1:1000 1:600 1:8000 1:800

SMI-31 SMI-311

Extensively phosphorylated neurofilaments Pan-neuronal marker

1:7500 1:1000

Drs. J. Whitson and C. Pike, IRU in Brain Aging, h'vine, CA Dr. Brian J. Cummings, IRU in Brain Aging, lrvine, CA Dakopatts, Glostrup, Denmark Seikagaku Kogyo Co. Ltd., Tokyo, Japan Upstate Biotechnology, Inc., Lake Placid, NY Boehfinger Mannheirn Corp., Indianapolis, IN Dr. Sharon G. Greenberg, Dementia Research, Burke Medical Res., White Plains, NY Sternberger Monoclonals Inc., Baltimore, MD Sternberger Monoclonals Inc., Baltimore, MD

1328 1342

Used to Detect

preincubation buffer for 5 min. Sections were again washed (5 x) in PB and mounted on gelatin coated slides. All sections were dehydrated through a graded series of alcohols and coverslipped with DePex (BHD, England). Immunohistochemistry controls utilized incubation with preabsorbed antisera (30 ~g antigen/Izt of prediluted serum overnight), incubation in buffer that did not contain primary antibody, or incubation with secondary antibodies to an incorrect species. The 13-amyloid antibodies detect synthetic 1-28 and 1-42 peptide on Western blots and are capable of detecting less than 10 ng of peptide with the avidin-biotin amplification as determined by dot blots (not shown). All immunohistochemical controls and preabsorptions were uniformly negative. Sections were examined and photographed on an Olympus BH-2 microscope.

Neuron and 13-Amyloid Quantification Nissl-positive neurons were counted by an observer blind to the extent of 13-amyloid accumulation using a 10 × 10 counting grid and 20× objective. Six random fields of entorhinal cortex were chosen from two sections per canine to reduce the effect of variance in section thickness. All numbers were pooled. Five random fields of entorhinal cortex from a neighboring section that had been pretreated with formic acid (5 min) and immunostained with 1342 (1:1000) were captured with a video microscope. These images were collected without digital filtering and saved to disk as grey scale images (256 levels). To avoid inaccurate quantification of staining density due to differences in tissue fixation, incubation variabilities, or other parameters that affect immunohistochemistry, we chose to quantify staining Area rather than staining Density. Images were binarized using a cutoff filter set at 150 grey levels and Image-1 software. In some cases, this cutoff may have omitted some very light 13-amyloid immunostaining but also eliminated accidental counting of background. The percentage values reported are, therefore, a conservative estimate. A 300 pixel by 300 pixel region centered on the screen was used to determine the percentage of pixels with values less than 150 (positive staining) in five fields of entorhinal cortex. Values were pooled to arrive at an estimate of the percentage area of entorhinal cortex immunopositive for 13-amyloid. RESULTS

Classical Markers for Pathology in Canine Brain We used a battery of immunohistochemical and histological stains on canine tissue to determine the types of plaques present within the canine brain. Bielschowsky-positive plaques were de-

tectable in four of eight beagle brains as well as the black Labrador. These Bielschowsky-positive plaques were moderately to lightly argentophilic. They appeared as even, granular patches of brown material on a yellow background. Rarely, a Bielschowskypositive plaque contained a few fine, dark fibers (Fig. IA). These fibers did not exhibit morphological indications characteristic of dystrophic neurites in the AD brain, i.e., swelling, tortuous growth patterns, or bulbous endings (Fig. 1B). In the canine brain, the diffuse Bielschowsky-positive plaques were detected throughout the cortex and hippocampal formation. No dystrophic fibers or neurofibrillary tangles were detected with Bielschowsky staining in any of the canine brains we examined, regardless of age, extent of 13-amyloid accumulation, or presence of plaque pathology. Upon staining with thioflavine S or Congo red, other routine histological stains for Alzheimer's-like pathology, plaques were not detected, even though adjacent sections clearly contained plaques revealed by 13-amyloid immunohistochemistry or Bielschowsky staining. However, we did observe amyloid angiopathy with Congo red staining. Thioflavine S-positive blood vessels were also observed, but were difficult to verify due to high levels of autofluorescence, possibly induced by long-term storage in fixative (see Fig. 2).

13-Amyloid Accumulation in Canine Brain Immunocytochemistry with antibodies to 13-amyloid (both 1328 and 1342) revealed a greater amount of pathology in the canine brains than the modified Bielschowsky stain. This increased detection of pathology was even more apparent when tissue sections were pretreated with 88% formic acid for 5 min. There were no obvious differences between 1328 and 1342 immunostaining. Formic acid pretreatment and irnmunohistochemical detection with 13-amyloid antibodies indicated the presence of plaques in seven of nine animals, two more than detected by modified Bielschowsky silver staining. 13-Amyloid immunostaining revealed extensive amyloid accumulation. Many plaques in the canine brain were significantly larger than those typically seen in the human brain (compare Fig. 1C and D). 13-Amyloid immunoreactivity was often diffusely spread throughout all layers of cortex except layer I. In one animal, approximately 20% of the total area of entorhinal cortex was occupied by 13-amyloid immunoreactivity (see Fig. 2A and below).

Intact Neurons Within Canine Plaques There were three unusual findings observed with 13-amyloid immunostaining. The first was the presence of numerous apparent

550

CUMMINGS ET AL.

D

FIG. 1. (A) High-power photo of Bielschowsky silver-positive plaque in the CA1 region of the dog. Note the rare, fine, dark, fiber (arrows) within this plaque compared to the thickened, twisted, dystrophic fibers found in human neuritic plaques. (B) Same magnification of Bielschowsky silver-positive plaque in region CA1 of the human brain. Numerous classical dystrophic neurites can be seen entering this plaque (arrows). (C) Low-power field of several 13-amyloid immunopositive plaques within the entorhinal cortex of the dog brain. Some accumulations of amyloid are greater than 300 I~m in diameter. Compare the size of these plaques to those from a human brain. (D) Same-power field as C of [3-amyloid immunopositive plaques within the human entorhinal cortex. Scale bars A and B = 40 Ixm; C and D = 100 p.m.

holes within canine plaques that did not stain positively for 13-amyloid. Many 13-amyloid-positive plaques were diffusely but strongly stained for 13-amyloid, yet contained spherical regions with no 13-amyloid staining within them. These holes within plaques were most evident in the entorhinal cortex and relatively rare in the dentate gyrus. Often, a large entorhinal plaque would have 10 or 20 holes within it, each approximately 10-15 ~m in diameter. The appearance of these holes within the plaques is consistent with the presence of intact cells there. We photographed these 13-amyloid-positive plaques, removed the coverslips, counterstained them with cresyl violet, and relocated the same plaques. We found that most of the holes were, indeed, cells (Fig. 3A and B) because they were filled with Nissl-positive cytoplasm and often contained visible, intact nuclei. Upon close examination, many of these intraplaque ceils appeared to be morphologically indistinguishable from neighboring cells outside of plaques, but

many were obscured by amyloid staining. These Nissl-filled holes were verified to be neurons and not glia by double-label immunohistochemistry because they stained positively with the panneuronal marker, SMI-311 (Fig. 3C). In some plaques where lighter t3-amyloid staining did not obscure the neurons, SMI-311positive dendrites emanating from these neurons were also detectable. The second unusual finding was 13-amyloid immunoreactivity within neurons and neuronal processes. We observed the accumulation of ~-amyloid-immunoreactivity within the cytoplasm, apical dendrites, and processes of some pyramidal neurons in CA I and entorhinal cortex (Fig. 3D). This was especially evident using a three-dimensional microscope (Edge Scientific Instrument Corporation, CA) not yet commercially available. In addition, faint 13-amyloid immunoreactivity appeared to accumulate in the neuropil around these 13-amyloid-immunoreactive neurons. Neuronal

AMYLOID ACCUMULATION IN AGED CANINE BRAIN

551

B

FIG. 2. (A) Low-power field of entorhinal cortex demonstrating the extensive amount of 13-amyloidaccumulation in the dog brain. These plaques were often larger than plaques in the AD brain and were found in all layers of entorhinal cortex except layer I. (B) Field (10x) of Congo red fluorescence. Visualization of Congo red staining with a rhodamine filter set is more sensitive than cross-polarization birefringence (37). Note that no Congo red-positive plaques are visible. C: Field (10x) in adjacent section of 13-amyloidimmunoreactivity within numerous plaques. (D) Field (10x) in adjacent section of thioflavine S fluorescence. Note that no thioflavine S-positive plaques are visible. Scale bars A = 600 p,m; B, C, and D = 150 ~xm.

processes immunoreactive for 13-amyloid were the only abnormal fibers detected within the canine brain, but did not exhibit classical indications of dystrophy (Fig. 3E and F).

~-Amyloid Accumulation in the Dentate Gyrus The third unusual finding seen in the canine brain was a concentrated 13-amyloid-immunopositive laminar gradient occupying the outer two-thirds of the molecular layer of the dentate gyrus and often the perforant path terminal regions of stratum-lacunosum moleculare of CA1 as well (Fig. 4A). This intense zone of 13-amyloid accumulation was present in four of the six 13-amyloidcontaining beagle brains. The molecular layer band was faintly visible in the black Labrador as well. In some cases, the molecular layer zone extended the complete length of the molecular layer paralleling the granule cells. In other cases, the zone only extended 1/2 or 1/3 the length of the dentate gyms. Similar intense

concentrations of 13-amyloid were not seen in the AD brain. However, the middle region of the molecular layer in the canine brain also often contained plaques arranged in a linear fashion, similar to those seen in humans. These dentate plaques were superimposed upon the concentrated molecular layer zone of 13-amyloid and rarely contained holes (Fig. 4B).

Neuronal Loss Does Not Correlate With f3-Amyloid Accumulation Due to the nature of the DOE study, no kennel-mates were sacrificed at early to middle time points. Therefore, we could not determine if cell loss occured with age in these animals. We did evaluate if there was a relationship between the amount of [3-amyloid accumulation and neuron number in neighboring sections. Counts of neurons from random fields of entorhinal cortex were compared with the area of [3-amyloid immunostaining in entorhi-

552

C U M M I N G S ET AL.

FIG. 3. A: High-power field of 13-amyloid-positive plaque from a dog brain. Note the "holes" or absence of 13-amyloid immunoreactivity within this plaque (arrowheads) and compare it to the human plaque in Fig. ID. (B) The same plaque as in A, counterstained with cresyl violet. Note the Nissl-positive ceils (arrowheads) within many of the holes in A. Not all holes contained Nissl-positive cells (arrows) (C) Pan neuronal marker (SMI-311) immunostained neurons (blue) which are superimposed within the [3-amyloid immunopositive plaque (brown). Several neurons that are indistinguishable from neurons within the surrounding neuropil are visible (arrows) (D) Localization of [3-amyloid immunoreactivity (brown) within the apical and basal dendrites (arrowheads) of these entorhinal cortical neurons counterstained with cresyl violet. (E,F) Left and right stereo images revealing the diffuse accumulation of 13-amyloid immunoreactivity within the apical dendrite of this intact pyramidal neuron (arrowhead). An amyloid filled fiber is nearby. This image was taken with a real time three-dimensional microscope provided by Edge Scientific Instrument Corporation, Los Angeles, CA. Scale bars A through D = 50 p,m; E and F = 30 lxm.

AMYLOID A C C U M U L A T I O N IN AGED CANINE BRAIN

B

553

I

FIG. 4. (A) Low-power field of fl-amyloid immunoreactive zone (arrows) corresponding to the terminal fields of the perforant path. This section was counterstained with cresyl violet. The area of amyloid accumulation runs parallel to the dentate gyrus granule cells along the middle to outer zone of the molecular layer and in stratum lacunosum moleculare of CA 1. (B) High-power field of plaques superimposed upon the 13-amyloidzone within the molecular layer. DG, dentate gyrus granule cells; HI, hilus; IM, inner molecular layer; OM, outer molecular layer. Scale bars A = 1000 p.m; B = 400 p.m. nal cortex from neighboring sections. This analysis revealed a positive correlation (r = 0.57) between neuron number and extent of 13-amyloid accumulation (Fig. 5). This does not indicate an expected decrease in neuron number in relationship to greater 13-amyloid accumulation. The animal with largest accumulation of ~3-amyloid in the entorhinal cortex was also the animal with the highest neuron count. When this animal is excluded from the correlation, r = 0.31. When animals were pooled into groups with 13-amyloid accumulation over greater than 2% of the area of en-

torhinal cortex (n = 5) and those without 13-amyloid accumulation ( < 2 % , n = 4), a Student's unpaired t-test indicated no significant difference between the number of neurons in these two groups (p --- 0.84).

Neuritic and Glial Markers We also examined the canine brains for dystrophic neurites and neurofibrillary tangles using antibodies previously demonstrated to

554

CUMMINGS ET AL.

Accumulation of B-Amylold Immunoreactivity versus Neuron Number

Percentage Amyloid Neuron Number

SO. m

400

.I

SO

s0o

10

I00

--=~--

ROOM54

ROOM55

ROOX42

ROOF48

DOOFB4

ROOY41

ROOY39

m

m

ROOMS0 Black Lab

Individual Dogs FIG. 5. Quantification of neuron number and area of 13-amyloid immunoreactivity within the entorhinal cortex in nine aged dogs. Neurons were counted in six fields and area of 13-amyloid was quantified in five fields using Image-1 software (see text for details). There is no clear relationship between amount of 13-amyloid accumulation and neuron number. detect these pathologies in AD: tau-1, PHF-1, or SMI-31. Immunostaining with these antibodies did not demonstrate either dystrophic neurites or neurofibrillary tangles. Similarly, upon immunostaining with antibodies to GFAP we did not observe the reactive astrocytes commonly associated with and/or infiltrating plaques in the AD brain. GFAP immunoreactivity was scattered throughout the canine brain but not specifically localized to plaques. Neurite Promoters Were Not Found Within Canine Plaques Because basic fibroblast growth factor (bFGF) (17,46) and heparan sulfate glycosaminoglycans (HSGAGs) (44,48) are associated with plaques in AD, we sought to determine if these substances are also present in the 13-amyloid deposits of the canine brain. We were unable to detect either bFGF- or HSGAG-positive plaques within any of the canine brains, even after formic acid pretreatment, bFGF antibodies did, however, weakly detect norreal-appearing neurons in all the canine brains, which were similar in appearance to bFGF-positive neurons we have reported in human brain (17). While HSGAG antibodies occasionally detected cell nuclei similar to those seen in the normal human brain, no HSGAG positive plaques or intensely labeled nuclei were seen in

the canine brain, even when we increased the concentration of both bFGF and HSGAG primary antibodies (Fig. 6A-C). Plaques in Human Cerebellum Plaques in the human cerebellum are generally of the diffuse to primitive subtype and do not contain dystrophic neurites. Therefore, plaques in human cerebellar tissue may be comparable to canine plaques. The cerebelli from five AD and two aged human controls were examined for plaques with a modified Bielschowsky silver stain and for 13-amyloid-immunoreactivity as well as bFGF, HSGAG, and SMI-31 or tau-1 immunostaining. Bielschowsky silver stain strongly revealed the presence of vascular amyloid accumulation and occasionally, weak, small plaque-like deposits in the cerebelli of two of the five AD cases. No plaques were detected in either of the control brains. None of the plaque-like structures detected within the AD cerebelli contained dystrophic neurites, although neuritic plaques were present within the hippocampal formation of the AD brains examined. When immunohistochemistry was utilized for f3-amyloid, bFGF and HSGAG detection in adjacent sections, numerous amyloidotic vessels were detected, as well as occasional 13-amyloid positive deposits (Fig. 6D-F). No bFGF or HSGAG-positive deposits were colocalized to

Canine

Human

FIG. 6. (A-C) Serial sections from canine and human (D-F) showing HSGAG, I~-amyloid, and bFGF immunostaining. Higher concentrations of primary antibodies were used in these series to insure faint pathology was not missed; therefore, background staining for HSGAG and bFGF were higher than normal. (A) HSGAG immunostaining in entorhinal cortex of the dog. Faint cytoplasm of cortical neurons is visible. (B) [3-Amyloid immunostaining in adjacent section to A demonstrates numerous plaques in this region. (C) bFGF immunostaining reveals moderate staining of cytoplasm, similar to that seen in normal human cortex. Neither bFGF nor HSGAG are strongly colocalized with t~-amyloid pathology. Arrowhead indicates region of blood vessel present throughout sections A, B, and C, (D) HSGAG immunostaining in the human AD cerebellum. Moderate staining of cell nuclei is visible. Purkinje cells are generally not HSGAG positive. (E) I~-Amyloid immunostaining in adjacent section to D demonstrates a moderate m o u n t of ~-amyloid-positive pathology including amyloid angiopathy. (F) bFGF immunostaining does not reveal any pathology, but does stain some cellular nuclei and the Purkinje cells of the cerebellum. Arrowheads indicate matching regions in adjacent sections. Scale bars for all photos -- 100 I.~m.

556

CUMMINGS ET AL.

regions of 13-amyloid immunoreactivity, even in formic acid pretreated sections or in tissue sections where the primary antibody was used at higher concentrations than rlormal. HSGAG immunoreactivity was seen within cellular nuclei as has previously been reported. In addition, bFGF immunoreacti~,ity was found in many cells, including Purkinje neurons. Neither tau-1 nor SMI-31, markers of dystrophic neurites, revealed any noticeable degeneration or abnormal processes in the human cerebelli examined (not shown). DISCUSSION Using aged brains from eight well-characterized beagles that received regular veterinary care and one black Labrador, we have extended the findings of Braunmiihl, (4), S61tysiak (45), Giaccone (16), Wisniewski (54), and others, who observed AD-like pathology in the aged canine brain. We report several new findings, including laminar extracellular concentrations of 13-amyloid accumulation within the terminal regions of the perforant path (i.e., molecular layer of the dentate gyrus and stratum-lacunosum moleculare of CA 1); the presence of numerous intact neurofilamentimmunopositive neurons within plaques; a lack of positive correlation between neuronal cell loss and diffuse 13-amyloid accumulation; the accumulation of amyloid-positive material within neurons and neurites; and the apparent absence of bFGF and HSGAG immunoreactivity within canine plaques. These findings are consistent with the prevalence of diffuse plaques and the general absence of dystrophic neurites within the canine brains examined here.

Majority of Plaques in These Canine Brains are Diffuse In the past, researchers may have referred to the AD-like pathology found in canine brains as senile or neuritic plaques because more specific nomenclature for plaque subtypes had not been established. In 1973, Wisniewski and Terry wrote an extensive review on the pathogenesis of the senile plaque (56). Their proposed plaque nomenclature, which is often used today, consists of three plaque subtypes (in chronological order): the primitive plaque, the classic plaque (also called a neuritic plaque), and the compact plaque. Using antibodies unavailable in 1973 or more sensitive modified silver stains, researchers have detected a new, earlier plaque type, consisting of non-B-pleated amyloid protein which is, thus, not thioflavine S-positive or Congophilic. This subtype has been called very primitive (13,52) or diffuse (21), and the amyloid present is thought to later aggregate into the 13-pleated sheet structure of I~-amyloid found in the primitive plaque (15,49). Over time, primitive plaques are thought to further evolve into neuritic plaques, which are characterized by the extensive involvement of degenerating or dystrophic neurites as well as glial involvement associated with a 13-amyloid core. When neuritic and glial elements have largely dissipated, the plaque is called a burnt out or compact plaque. There is still confusion as to nomenclature (9); e.g., whether neuritic plaques are surrounded by astrocytes more than compact plaques or when dystrophic neurites arise (25,27). However, for the purposes of this report, we refer to plaques as diffuse (i.e., non-13-pleated), primitive, neuritic, and compact in a presumed chronological progression. We did not detect any thioflavine S- or Congo red-positive plaques in the eight beagles we examined. The inability to detect positive staining for thioflavine S or Congo red in these canines suggests that the amyloid deposits present are at an early stage of development, similar to the diffuse stage plaque in AD, and that the amyloid has not yet assumed a 13-pleated sheet conformation. Others have suggested that neuritic plaques are present within the canine brain. Based on previous reports and our observations (41),

however, their occurrence appears to be rare. Previously, Selkoe et al. reported thioflavine S-positive amyloid deposits in the cortical capillaries and meningeal arteries in all nine aged canines examined but thioflavine S-positive plaques in only five of the canines (42). In addition, Pauli et al. photographically demonstrated strong thioflavine S-positive plaques in dog brain (31). Such findings indicate that under appropriate conditions, plaque development in some canine may proceed to a more advanced level. It is also possible that prolonged fixation may have altered the detectability of plaques. In general, however, the majority of plaques reported in the canine brain by other researchers are diffuse and not thioflavine Sor Congo red-positive. For example, while Uchida et al. indicated senile or classical plaques are present within the canine brain, this subtype was only detected in 1 dog out of 90 examined; nearly all the plaques in these brains were actually classified as diffuse or primitive (51 ). Wisniewski and Terry have also referred to neurites associated with some canines plaques, but the processes they detected were likely not dystrophic because they stated there was ++a lack of neuritic reaction to the amyloid" and that canine plaques "lacked the twisted tubules of AD plaques" (54).

Cerebral Arnyloid Angiopathy in the Aged Canine Our observations confirm the findings of others that thioflavine S- and Congo red-positive changes are present within the vasculature of aged dogs. Most researchers, including ourselves, have found cerebral amyloid angiopathy in canine brain using either thioflavine S or Congo red staining (42,45,50,51,54). We found evidence of amyloid angiopathy in eight of nine dogs (Table 3). Uchida et al. have studied amyloid angiopathy in dogs extensively (50,51). In their largest study, cerebral amyloid angiopathy was detected in 31% of 90 dogs ranging in age from 0 to 19 years. They found that the incidence of amyloid angiopathy in dogs increases with age, reaching 100% by age 15. In some cases, amyloid deposition could be found within the submucosa connective tissue of the intestine, the left artrioventricular valve, and arteries of the lung and liver (51). Interestingly, the one beagle in our study that did not have detectable amyloid angiopathy was 17 years old, while the beagle with only a few vessels affected was 13 years old (Table 3). Unfortunately, we did not have access to TABLE 3 DOE ID NUMBERSOF RESPECTIVEDOGS AND THE SEMIQUANTITATIVELEVELOF 13-AMYLOID IMMUNOSTAININGOBSERVED DOE ID CA ROOM54 ROOM55 ROOX42 ROOF48 DOOF24 ROOY41 ROOY39

ROOM50 Black Lab

Amyloid Angiopathy

Plaques

Molecular LayerZone

+ + ++ 0 ++ + + ++

++ ++ ++ ++ ++ 0

++ ++ + + ++ 0 0

+

+

O

+ + +

0 ++

0 +

For amytoid angiopathy and number of plaques, + + indicates numerous vessels or plaques were detected, + indicates a few vessels or rare plaques were detected, and 0 indicates none. For 13-amyloidpresence in the molecular layer of the dentate gyms, + + indicates a moderate to strong concentration of 13-amyloid, + indicates a faint concentration, and 0 indicates no 13-amyloid was detectable.

AMYLOID ACCUMULATION IN AGED CANINE BRAIN non-CNS tissue. In humans, as well as canines, age-related amyloidosis occurs within the cerebral arterioles and capillaries of the neocortex as well as within other organs, such as the heart, intestines, and skin (20,51). Thus, in addition to canines being a model for early plaque formation, aged canines are an excellent model for amyloid angiopathy.

Neuronal Processes With the Aged Canine Brain Appear Normal We did not find clear evidence of dystrophic neurites or NFTs in the aged canine brains examined using antibodies to tau-l, PHF-1, or SMI-31. We did see rare Bielschowsky-positive fibers in a few canine plaques, but their contour was fine and regular. Thus, based upon both their appearance (see Fig. 1A,B) and lack of supporting data from immunohistochemical observations, we did not classify these Bielschowsky-positive fibers as dystrophic. While overfixation may have reduced the ability to detect early neuritic changes, Ulrich et al. support the position that neuronal processes within the aged dog are normal. They detected normal neurons in dog brains stained with antibodies to paired helical filaments (PHF) but did not find PHF assembled into the abnormal fibers of neurofibrillary tangles found in AD (53). Wisniewski and Terry did not observe neurofibrillary tangles in the dog brain either (54). As with nonhuman primates, it appears that canines will not make a good model for NFT formation. It is possible that NFr changes occur later in the disease, or that their antigenicity is sufficiently different that human NFT markers cannot detect changes in these tissues.

Lack of Trophic Factors and GliaI Involvement Within Beagle Plaques In agreement with previous reports, we did not see an accumulation of glial cells around the amyloid containing lesions in the canine brains examined based on their Nissl staining characteristics. These observations were verified in additional sections by GFAP immunostaining for astrocytes. This is consistent with our suggestion that the plaques seen in the canine brain are the earliest form of plaque and have yet to attract astrocytes, microglia, and neurites. We suggested previously that the bFGF and HSGAGs found within many human plaques may be produced by surrounding glia or injured neurons in the region (17,48), and that these molecules may be responsible for attracting neurites into plaques (8,11). In the present study, we were unable to detect bFGF immunoreactivity colocalized with canine plaques, possibly because there is little glial involvement or because these are early plaques. It is also possible that suboptimal fixation reduced our ability to detect bFGF and/or HSGAGs, and further studies with fresh tissue are warranted. Snow and Wight have argued that one role for HSGAGs in the formation of plaques is to assist in the assembly of 13-amyloid into 13-pleated sheets [for review see (44)]. The observation that HSGAGs are not detectable within these canine plaques and that these plaques are not thioflavine S- or Congo red-positive supports this position.

Plaques in the Cerebellum of AD Cases Show Features Similar to Canine Plaques Plaques in the cerebellum of AD cases with known neuritic involvement in the cortex were examined. There is general agreement that the plaques found within the cerebellum are mostly of the diffuse subtype, although amyloid angiopathy is also found there. If these cerebellar plaques are nonneuritic, we would predict that trophic factors and substrate molecules would not be present.

557 Using Bielschowsky silver staining and antibodies to [3-amyloid, we observed amyloid angiopathy and amyloid accumulation within the AD cerebellum, but we did not observe dystrophic neurites. In adjacent sections to those stained with 13-amyloid antibodies, we were also unable to detect colocalization of abnormal accumulations of either bFGF- or HSGAG-immunoreactivity with the [3-amyloid deposition.

Relationship Between Neurons and [3-Amyloid Accumulation We did not observe a clear relationship between neuron number and amount of deposited 13-amyloid. If 13-amyloid is neurotoxic, one would predict a negative correlation between neuron number and amyloid accumulation. We found a weak positive correlation (r = 0.57). This is consistent with our finding 13-amyloid accumulation within neurons; the more neurons present, the greater the amyloid production. It is also possible that, in these aged brains, neuron loss has already occurred at this late stage and, thus, would not correlate with 13-amyloid accumulation. For valid quantification across animals it is important that all animals be raised in the same stable environment to reduce variability (5). This may explain why both S61tysiak (45) and Wisniewski (54) found cell loss with age in canines from several breeds. While it would be useful to compare the neuron numbers here to younger beagles to confirm if there was generalized cell loss with age, this type of tissue was unavailable. In addition, due to the nature of the tissue source, we were unable to control for variations in tissue shrinkage across animals. Because there was no clear trend between neuron number an amyloid accumulation, this likely did not affect our analysis. However, the absolute neuron numbers shown in Fig. 5 should be considered approximate. Neurons appeared to be within the center of most, but not all diffuse plaques. We found apparently normal, SMI-311 immunoreactive neurons within the majority of plaques detected with [3-amyloid antibodies in the canine brain (see Fig. 3C). Plaques within the dentate gyms, however, rarely contained holes of no 13-amyloid immunoreactivity and also did not contain neurons immunopositive for SMI-311. Giaccone et al. also showed an example of neurons within canine plaques (their Fig. I) but do not comment on this observation in the text (16). As they did not find any evidence of PHF-mediated degeneration with silver staining or antibodies to PHF and Alz-50, they, too, conclude that these preamyloid deposits precede neuritic involvement. Interestingly, the presence of intact neurons within some human plaques that appear similar to those we show in canines has also been reported (3).

[3-Amyloid Accumulation Within Neurons We have suggested that in the human brain, the accumulation of 13-APP within degenerating neurons precedes plaque formation in the majority of cases (12). Interestingly, we saw the accumulation of 13-amyloid reaction product within the apical and basal processes of intact neurons in the dog brain (see Fig. 3D). The nuclear zone was clear of reactivity, and at this level of analysis, these neurons appeared normal. There may be a parallel between this observation and Perry et al.'s observation that far more neurofibrillary tangles in the human brain axe [3-arnyloid immunoreactive than previously believed (32). Thus neurons in both the human and dog may accumulate 13-amyloid. Coupled with recent data that many cell types produce 13-amyloid normally in culture (19), and that human mixed-brain cell cultures also release 13-amyloid (43), it is possible that these processes are more visible in canine tissue. While canine neurons were not labeled with conventional markers for NFrs, an abnormality is likely occurring within these [3-amyloid-positive neurons, as most canine neurons

558

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are not 13-amyloid positive. Further in vivo research to characterize this process is necessary. That canine neurons may be entering an early stage of NFT development which is not yet detectable by conventional markers is particularly interesting, given the zone of 13-amyloid accumulation within the terminal fields of the perforant path. Koo et al. recently showed that 13-APP undergoes fast anterograde axonal transport and suggested that 13-amyloid deposition may be the result of aberrant processing of 13-APP within neurite terminals (23). Both the middle and outer molecular layer of the dentate gyrus and, in some cases, stratum-lacunosum moleculare of CA1, were regions with extensive 13-amyloid accumulation, further suggesting that canine entorhinal neurons are overproducing 13-amyloid. Factors Contributing to 13-Amyloid Accumulation In vitro studies have shown that 13-amyloid can be neurotrophic or neurotoxic, depending on its aggregation state or other conditions. The effect of [3-amyloid on cultured neurons appears to be dependent upon the assembly state of the 13-amyloid--which is affected by its amino acid sequence--and the viability of neurons. Only following the self-assembly of amyloid into thioflavine S-positive aggregates does the substance induce neuritic dystrophy and neuronal death in vitro (33-36). In this state, 13-amyloid places select neurons at risk for degeneration (e.g., glutamate mediated excitotoxicity or apoptosis), interestingly, in cell culture not all neurons exposed to aggregated 13-amyloid degenerate over the course of the experiment (33). In AD tissue, diffuse plaques are not associated with degenerating neurites and, thus, one or more factors that contribute to cell loss are yet undeveloped, e.g., the particular assembly state of amyloid or the absence of neurite attracting molecules. Consistent with this idea, in Dutch amyloidosis, diffuse amyloid plaques are commonly found but neuritic plaques are not observed (7,18). In addition, in Down's syndrome, neuronal dystrophy appears to coincide with the transition of 13-amyloid into thioflavine S- and Congo red-positive structures. Prior to this stage, Down's syndrome plaques do not contain dystrophic neurites (15). Others have also stated that in AD, dystrophic neurites do not appear until plaques become Congophilic (40). It has also been suggested that the extent of neuritic dystrophy within plaques and not the number of plaques per se correlates strongest with clinical signs of dementia (28). Thus, our finding of neurons within canine plaques may be compatible with in vitro data that suggest that the neurotoxicity of 13-amyloid is related to its formation of thioflavine S- and/or Congo red-positive aggregates (33,36). In addition, it is not yet known whether the 13-amyloid in the canine brain contains the same amino acid sequence as found in AD. It is possible that a longer, nontoxic fragment is present that still contains the 1328

and 1342 antibody recognition sites. It should also be pointed out that in vitro data may not represent the full range of relevant variables to the in vivo condition and caution is warranted. Because some researchers have observed neuritic plaques within the canine brain, it must be possible for canine plaques to evolve into more advanced stages. As has been suggested for AD pathology, risk factors may promote plaque development in the dog into aggregated thioflavine S- or Congo red-positive stages. This may be dependent upon other risk factors such as the animal's age, breed, health, (e. g., head trauma, hypertension, ischemia) or environmental factors. S61tysiak suggested that dogs raised in an industrial environment exhibited greater pathology than dogs from a rural environment (45). The absence of advanced plaque development in our study may be the result of a controlled living environment, be due to a common genetic background (4I), or because other elements were not present within the plaques. Model of Plaque Formation Initially, 13-amyloid appears to accumulate diffusely in the neuropil and within neurons. We have shown that 13-amyloid collects as an immunoreactive laminar zone corresponding to the terminal field of the perforant path, particularly within the molecular layer of the dentate gyms. This is the same region where plaques form a laminar pattern in the human brain. As the concentration of 13-amyloid increases, it may condense into plaque-like structures. Over time, 13-amyloid forms a 13-pleated sheet structure which is thioflavine S- and Congo red-positive and is termed a primitive plaque. We did not detect these later stage plaques in these canine brains. Thus, the conformational state of 13-amyloid in canine plaques may not be directly neurotoxic. Conversely, the 13-amyloid peptide sequence in the canine may not be identical to the human case. Next, primitive plaques develop into neuritic plaques as dystrophic neurites and glia become involved. Our observations in canine tissue support the theory that trophic and/or substrate molecules may contribute to neuritic involvement (8,10) because we detected neither of these factors in these canine plaques nor dystrophic neurites. Finally, many canine plaques contain neurons and the accumulation of this diffuse 13-amyloid in these canine brains appears not to induce extensive cell loss. Thus, the aged canine brain appears to represent a model of early plaque development. This model may be useful for delineating the mechanisms involved in initial 13-amyloid deposition and studying the processes that promote plaque development into degenerative loci. ACKNOWLEDGEMENTS We would like to thank Drs. Jennifer Kahle and Christian Pike for helpful discussions and critical review of the manuscript as well as Dr. Jolanta Ulas and Anja Kammesheidt for translation of Polish and German manuscripts. This work was supported by a NIA "Aging Program Project" Grant AG 00538.

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